Galectin-3 ablation protects mice from diet-induced NASH: A major scavenging role for galectin-3 in liver Carla Iacobini 1,  , Stefano Menini 1,  , Carlo Ricci 1,  , Claudia Blasetti Fantauzzi 1 , Angela Scipioni 1 , Laura Salvi 1 , Samantha Cordone 1 , Francesca Delucchi 1 , Matteo Serino 2 , Massimo Federici 2 , Flavia Pricci 3 , Giuseppe Pugliese 1,⇑ 1 Department of Clinical Sciences, ‘‘La Sapienza’’ University, Rome, Italy; 2 Department of Internal Medicine, ‘‘TorVergata’’ University, Rome, Italy; 3 Department of Cell Biology and Neurosciences, National Institute of Health of Italy, Rome, Italy Background & Aims: Excess fatty acid oxidation and generation of reactive carbonyls with formation of advanced lipoxidation endproducts (ALEs) is involved in nonalcoholic steatohepatitis (NASH) by triggering inflammation, hepatocyte injury, and fibro- sis. This study aimed at verifying the hypothesis that ablation of the ALE-receptor galectin-3 prevents experimental NASH by reducing receptor-mediated ALE clearance and downstream events. Methods: Galectin-3-deficient (Lgals3 À/À ) and wild type (Lgals3 +/+ ) mice received an atherogenic diet or standard chow for 8 months. Liver tissue was analyzed for morphology, inflamma- tion, cell and matrix turnover, lipid metabolism, ALEs, and ALE- receptors. Results: Steatosis was significantly less pronounced in Lgals3 À/À than Lgals3 +/+ animals on atherogenic diet. NASH, invariably detected in Lgals3 +/+ mice, was observed, to a lower extent, only in 3/8 Lgals3 À/À mice, showing less inflammatory, degenerative, and fibrotic phenomena than Lgals3 +/+ mice. This was associated with higher circulating ALE levels and lower tissue ALE accumu- lation and expression of other ALE-receptors. Up-regulation of hepatic fatty acid synthesis and oxidation, inflammatory cell infiltration, pro-inflammatory cytokines, endoplasmic reticulum stress, hepatocyte apoptosis, myofibroblast transdifferentiation, and impaired Akt phosphorylation were also significantly attenu- ated in Lgals3 À/À animals. Galectin-3 silencing in liver endothelial cells resulted in reduced N e -carboxymethyllysine-modified albu- min uptake and ALE-receptor expression. Conclusions: Galectin-3 ablation protects from diet-induced NASH by decreasing hepatic ALE accumulation, with attenuation of inflammation, hepatocyte injury, and fibrosis. It also reduced up-regulation of lipid synthesis and oxidation causing less fat deposition, oxidative stress, and possibly insulin resistance. These data suggest that galectin-3 is a major receptor involved in ALE uptake by the liver. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction Non-alcoholic fatty liver disease (NAFLD) encompasses various disease conditions, from simple steatosis to nonalcoholic steato- hepatitis (NASH), cirrhosis, and possibly hepatocellular carci- noma [1]. It is associated with insulin resistance and the metabolic syndrome [2], which causes fat accumulation within the liver via increased fatty acid (FA) delivery from adipose tissue and enhanced hepatic FA import and synthesis exceeding the rate of FA export and catabolism [3]. Based on the ‘‘two hits’’ hypothesis, transition from steatosis to NASH is dependent on the superimposition of oxidative phe- nomena (‘‘second hit’’) on top of fat accumulation (‘‘first hit’’), which sensitizes the liver to oxidant-dependent injury resulting in tissue inflammation and hepatocyte degeneration [4]. More recently, it has been suggested that FA metabolism itself may be responsible for this transition [5], via induction of endoplas- mic reticulum (ER) stress and increased production of reactive oxygen species (ROS) by mitochondrial b-oxidation and, when its capacity becomes overwhelmed, also by peroxisomal b-oxida- tion and endoplasmic reticulum x-oxidation of FAs [4]. In turn, ROS would trigger the self-reinforcing inhibitor jB kinase-b/ nuclear factor jB (NFjB)/tumour necrosis factor-a (TNF)-a cycle [6], which initiates and maintains a T helper 1 (Th1)- and macro- phage 1 (M1)-mediated inflammatory response [7]. Further pro- gression to cirrhosis may require a ‘‘third hit’’, which specifically promotes fibrosis by inducing myofibroblast transdifferentiation of hepatic stellate cells (HSCs), a step involving a shift of the innate immune system toward a Th2- and M2-phenotype [7]. ROS also peroxidize unsaturated lipids generating endoperox- ides, which are further metabolized to reactive carbonyl species (RCS), such as 4-hydroxy-2-nonenal (HNE) and glyoxal. RCS and the advanced lipoxidation endproduct (ALE) adducts or cross- links, generated by their reaction with proteins [8], are more per- sistent than ROS, thus causing more inflammation and injury and also direct activation of fibrogenesis [9]. The liver is the main catabolic site for ALEs and advanced glyca- tion endproducts (AGEs), as indicated by the findings that plasma Journal of Hepatology 2011 vol. xxx j xxx–xxx Keywords: Steatosis; Inflammation; Fibrosis; Advanced lipoxidation endprod- ucts; Oxidative stress. Received 28 December 2009; received in revised form 8 September 2010; accepted 14 September 2010 ⇑ Corresponding author. Address: Department of Clinical Sciences, ‘‘La Sapienza’’ University of Rome, Viale del Policlinico, 155-00161 Rome, Italy. Tel.: +39 0633775049; fax: +39 0633775001. E-mail address: giuseppe.pugliese@uniroma1.it (G. Pugliese).   These contributed equally to this work. Research Article Please cite this article in press as: Iacobini C et al. Galectin-3 ablation protects mice from diet-induced NASH: A major scavenging role for galectin-3 in liver. J Hepatol (2011), doi:10.1016/j.jhep.2010.09.020